Mature manmalian neurons are incapable of cell division and cannot, with the exception of olfactory neurons, be generated from stem cells in the adult nervous system. Thus, continuous dividing clonal cell lines with neuronal characteristics have proven to be very useful to neurobiologists studying almost every aspect of the nervous system. Such cell lines allow the generation of large numbers of homogeneous cells and the manipulation of these cells through gene transfer to yield novel derivatives expressing foreign gens products. These advantages have led to the development and characterization of a variety of neuronal cell lines, some of which have been useful for cell biological, biochemical, and molecular biological studies. The utility of these different cell lines and their ability to approximate aspects of the neuronal phenotype vary widely. However, studies conducted over the past decade have shown that the usefulness of a cell line primarily depends on two characteristics: 1) the extent to which a particular cell line resembles post-mitotic neurons; and 2) the doubling time. Frequently, these two key characteristics are inversely related. Many differentiated properties of neurons are not fully articulated in vivo until the stem cell becomes post-mitotic. However, rapidly dividing neuronal cell lines usually do not possess the phenotypic properties of terminally differentiated non-dividing neurons, instead, they often resemble in vivo neuroblasts or embryonic neurons. For example, many of these cell lines elaborate immature neurites with an immature cytoskeleton but lack most of the morphology and neuritic differentiation of post-mitotic neurons. Nevertheless, since they divide rapidly, these cell lines are useful for biochemical and transfection experiments. Naturally occurring neoplastic derivatives of many neuronal cell types of the central (CNS) and peripheral (PNS) nervous systems usually fall in this category (e.g., neuroblastomas, pheochromocytomas and medulloblastomas). At the other end of the spectrum are cell lines exemplified by HCN 1 (Ronnett et al., 1990, Science, 248:603-605). These cells have many characteristics of differentiated neurons, but they divide so slowly (i.e., doubling time of 72 hours when undifferentiated) that they are not amenable to many experimental manipulations. Even PC 12 cells, the classic example of a neuronal cell line, revert to their less neuronal, rapidly dividing phenotype upon removal of NGF (Greene and Tischler, 1982, Advances in Cellular Neurobiology, S. Federoff and L. Hertz, eds., Academic Press, New York).
Recently, considerable effort has been expended to immortalize specific neuronal precursors that are found transiently during development (for recent reviews, see Cepko, Ann. Rev. Neuro., 12:47-65, 1989; or Lendahl and McKay, TINS, 13:132-137,1990; and, for specific examples see, Bartlett et al., Proc. Natl. Acad. Sci. USA, 85:3255- 3259, 1988; Fredericksen et al., Neuron, 1:439-448, 1988; Bitten et al., Neuron, 4:189-201 (1990); Hammang, et al, Neuron, 4:775-782, 1990; Ryder et al., J. Neurobiol., 21:356-375, 1990; Lo et al., Dev. Biol., 145:139-153, 1991). This approach is particularly valuable because these cell lines seem to approximate characteristics of specific cell types at particular stages of development. Already, new molecules which may serve important developmental functions have been isolated using these novel cell lines (Johnson et al., Nature, 346:858-861, 1990; Lendahl et al., Cell, 60:585-595, 1990). However, with the exception of MAH cells (Birren et al., Neuron 4: 189-201, 1990), cell lines generated using this strategy have a limited ability to undergo further neuronal differentiation. Rather, they seem to be more useful for examining specific branch points in the emergence of neuronal lineages.
The ideal cell line for analysis of the processes of neuronal maturation and the intrinsic factors which affect the establishment of the neuronal phenotype would be one that divides rapidly so that it could be grown in large quantities and transfected to produce a stable population of cells expressing exogenous gene products. Upon induction with an agent promoting differentiation, this ideal cell line would leave the cell cycle, undergo an irreversible commitment to a neuronal phenotype, and exist in a stable post-mitotic state. These cells would subsequently elaborate extensive neuritic processes and would mature to a state similar to that of primary neurons in culture.
Embryonal carcinoma cell lines satisfy some of the above criteria. These cells, which have been derived from both murine and human embryonal tumors, consist of undifferentiated multipotential cells which will differentiate into one or several cell types when placed under certain conditions (usually including treatment with retinoic acid [RA]). This process resembles the actual commitment to different phenotypes which are found in vivo. These cell types frequently include neurons, glial, muscle, and/or endothelial cells at various stages of differentiation. Thus, their usefulness to neurobiologists is limited by their heterogeneity. NTera 2/D1 (NT2), a human teratocarcinoma cell line, has characteristics in common with its murine counterparts in that they are capable of undergoing phenotypic changes in response to RA. However, unlike most of the murine embryonal carcinoma cell lines, the only identifiable phenotype found following RA treatment of NT2 cells are neurons (Andrews, Dev. Biol., 103:285-293 (1984); Andrews et al., Lab. Invest., 50:147-162 (1984); Lee and Andrews, J. Neurosci., 6:514-521 (1986). Unfortunately, in all previous studies, these neurons represented only a small percentage of the cells, and they coexisted with a large unidentified population of dividing large flat cells and a residual number of undifferentiated stem cells (Andrews, 1984).